Toluene permeabilization differentially affects F- and P-type ATPase activities present in the plasma membrane of Streptococcus mutans

Streptococcus mutans membrane-bound P- and F-type ATPases are responsible for H+ extrusion from the cytoplasm thus keeping intracellular pH appropriate for cell metabolism. Toluene-permeabilized bacterial cells have long been used to study total membrane-bound ATPase activity, and to compare the properties of ATPase in situ with those in membrane-rich fractions. The aim of the present research was to determine if toluene permeabilization can significantly modify the activity of membrane-bound ATPase of both F-type and P-type. ATPase activity was assayed discontinuously by measuring phosphate release from ATP as substrate. Treatment of S. mutans membrane fractions with toluene reduced total ATPase activity by approximately 80% and did not allow differentiation between F- and P-type ATPase activities by use of the standard inhibitors vanadate (3 µM) and oligomycin (4 µg/mL). Transmission electron microscopy shows that, after S. mutans cells permeabilization with toluene, bacterial cell wall and plasma membrane are severely injured, causing cytoplasmic leakage. As a consequence, loss of cell viability and disruption of H+ extrusion were observed. These data suggest that treatment of S. mutans with toluene is an efficient method for cell disruption, but care should be taken in the interpretation of ATPase activity when toluene-permeabilized cells are used, because results may not reflect the real P- and F-type ATPase activities present in intact cell membranes. The mild conditions used for the preparation of membrane fractions may be more suitable to study specific ATPase activity in the presence of biological agents, since this method preserves ATPase selectivity for standard inhibitors.

Visualization of a peripheral stalk in V-type ATPase: Evidence for the stator structure essential to rotational catalysis

F- and V-type ATPases are central enzymes in energy metabolism that couple synthesis or hydrolysis of ATP to the translocation of H+ or Na+ across biological membranes. They consist of a soluble headpiece that contains the catalytic sites and an integral membrane-bound part that conducts the ion flow. Energy coupling is thought to occur through the physical rotation of a stalk that connects the two parts of the enzyme complex. This mechanism implies that a stator-like structure prevents the rotation of the headpiece relative to the membrane-bound part. Such a structure has not been observed to date. Here, we report the projected structure of the V-type Na+-ATPase of Clostridium fervidus as determined by electron microscopy. Besides the central stalk, a second stalk of 130 Å in length is observed that connects the headpiece and membrane-bound part in the periphery of the complex. This additional stalk is likely to be the stator.

The zntA gene of Escherichia coli encodes a Zn(II)-translocating P-type ATPase

The first Zn(II)-translocating P-type ATPase has been identified as the product of o732, a potential gene identified in the sequencing of the Escherichia coli genome. This gene, termed zntA, was disrupted by insertion of a kanamycin gene through homologous recombination. The mutant strain exhibited hypersensitivity to zinc and cadmium salts but not salts of other metals, suggesting a role in zinc homeostasis in E. coli. Everted membrane vesicles from a wild-type strain accumulated 65Zn(II) and 109Cd(II) by using ATP as an energy source. Transport was sensitive to vanadate, an inhibitor of P-type ATPases. Membrane vesicles from the zntA∷kan strain did not accumulate those metal ions. Both the sensitive phenotype and transport defect of the mutant were complemented by expression of zntA on a plasmid.

Crystal structure of the regulatory subunit H of the V-type ATPase of Saccharomyces cerevisiae

In contrast to the F-type ATPases, which use a proton gradient to generate ATP, the V-type enzymes use ATP to actively transport protons into organelles and extracellular compartments. We describe here the structure of the H-subunit (also called Vma13p) of the yeast enzyme. This is the first structure of any component of a V-type ATPase. The H-subunit is not required for assembly but plays an essential regulatory role. Despite the lack of any apparent sequence homology the structure contains five motifs similar to the so-called HEAT or armadillo repeats seen in the importins. A groove, which is occupied in the importins by the peptide that targets proteins for import into the nucleus, is occupied here by the 10 amino-terminal residues of subunit H itself. The structural similarity suggests how subunit H may interact with the ATPase itself or with other proteins. A cleft between the amino- and carboxyl-terminal domains also suggests another possible site of interaction with other factors.

A heavy metal P-type ATPase OsHMA4 prevents copper accumulation in rice grain

Acknowledgements We thank B. Lahner, E. Yakubova and S. Rikiishi for ICP-MS analysis, N. Komiyama, Iowa State University Plant Transformation Facility and Prashant Hosmani for generation of transgenic rice, K. Wang for providing pTF101.1 vector and N. Verbruggen for providing pYES2 and pYEC2/CT-GFP vectors. We also thank Rice T-DNA Insertion Sequence Database center for providing the T-DNA insertion line and X. Wang, T. Zheng and Z. Li for accessing 3 K rice genome sequence, and Graeme Paton for helpful discussions on Cu bioavailability in water-logged soils. This research was supported by a Grant-in-Aid for Specially promoted Research (JSPS KAKENHI Grant Number 16H06296 to J.F.M), and the US National Science Foundation, Plant Genome Research Program (Grant #IOS 0701119 to D.E.S., M.L.G. and S.R.M.P.).

Cation Transport Coupled to ATP Hydrolysis By the (Na, K)-ATPase AN INTEGRATED, ANIMATED MODEL

An Adobe (R) animation is presented for use in undergraduate Biochemistry courses, illustrating the mechanism of Na(+) and K(+) translocation coupled to ATP hydrolysis by the (Na, K)-ATPase, a P(2c)-type ATPase, or ATP-powered ion pump that actively translocates cations across plasma membranes. The enzyme is also known as an E(1)/E(2)-ATPase as it undergoes conformational changes between the E(1) and E(2) forms during the pumping cycle, altering the affinity and accessibility of the transmembrane ion-binding sites. The animation is based on Horisberger`s scheme that incorporates the most recent significant findings to have improved our understanding of the (Na, K)-ATPase structure function relationship. The movements of the various domains within the (Na, K)-ATPase alpha-subunit illustrate the conformational changes that occur during Na(+) and K(+) translocation across the membrane and emphasize involvement of the actuator, nucleotide, and phosphorylation domains, that is, the ""core engine"" of the pump, with respect to ATP binding, cation transport, and ADP and P(i) release.

Cation Transport Coupled to ATP Hydrolysis By the (Na, K)-ATPase : an integrated, animated model

An Adobe (R) animation is presented for use in undergraduate Biochemistry courses, illustrating the mechanism of Na+ and K+ translocation coupled to ATP hydrolysis by the (Na, K)-ATPase, a P-2c-type ATPase, or ATP-powered ion pump that actively translocates cations across plasma membranes. The enzyme is also known as an E-1/E-2-ATPase as it undergoes conformational changes between the E-1 and E-2 forms during the pumping cycle, altering the affinity and accessibility of the transmembrane ion-binding sites. The animation is based on Horisberger's scheme that incorporates the most recent significant findings to have improved our understanding of the (Na, K)-ATPase structure function relationship. The movements of the various domains within the (Na, K)-ATPase alpha-subunit illustrate the conformational changes that occur during Na+ and K+ translocation across the membrane and emphasize involvement of the actuator, nucleotide, and phosphorylation domains, that is, the "core engine" of the pump, with respect to ATP binding, cation transport, and ADP and P-i release.

Probing the SERCA1a sarcoplasmic reticulum Ca2+-ATPase phosphorylation-site mutant D351E with inorganic phosphate

The expression of sarcoplasmic reticulum SERCA1a Ca2+-ATPase wild-type and D351E mutants was optimized in yeast under the control of a galactose promoter. Fully active wild-type enzyme was recovered in yeast microsomal membrane fractions in sufficient amounts to permit a rapid and practical assay of ATP hydrolysis and phosphoenzyme formation from ATP or Pi. Mutant and wild-type Ca2+-ATPase were assayed for phosphorylation by Pi under conditions that are known to facilitate this reaction in the wild-type enzyme, including pH 6.0 or 7.0 at 25ºC in the presence of dimethylsulfoxide. Although glutamyl (E) and aspartyl (D) residue side chains differ by only one methylene group, no phosphoenzyme could be detected in the D351E mutant, even upon the addition of 40% dimethylsulfoxide and 1 mM 32Pi in the presence of 10 mM EGTA and 5 mM MgCl2. These results show that in the D351E mutant, increasing hydrophobicity of the site with inorganic solvent was not a sufficient factor for the required abstraction of water in the reaction of E351 with Pi to form a glutamylphosphate (P-E351) phosphoenzyme moiety. Mutation D351E may disrupt the proposed alignment of the reactive water molecule with the aspartylphosphate (P-D351) moiety in the phosphorylation site, which may be an essential alignment both in the forward reaction (hydrolysis of aspartylphosphate) and in the reverse reaction (abstraction of water upon formation of an aspartylphosphate intermediate).

Structure and metal binding studies of the second copper binding domain of the Menkes ATPase

Biological utilisation of copper requires that the metal, in its ionic forms, be meticulously transported, inserted into enzymes and regulatory proteins, and excess be excreted. To understand the trafficking process, it is crucial that the structures of the proteins involved in the varied processes be resolved. To investigate copper binding to a family of structurally related copper-binding proteins, we have characterised the second Menkes N-terminal domain (MNKr2). The structure, determined using H-1 and N-15 heteronuclear NMR, of the reduced form of MNKr2 has revealed two alpha-helices lying over a single beta-sheet and shows that the binding site, a Cys(X)(2)Cys pair, is located on an exposed loop. H-1-N-15 HSQC experiments demonstrate that binding of Cu(I) causes changes that are localised to conserved residues adjacent to the metal binding site. Residues in this area are important to the delivery of copper by the structurally related Cu(I) chaperones. Complementary site-directed mutagenesis of the adjacent residues has been used to probe the structural roles of conserved residues. (C) 2003 Published by Elsevier Inc.

Characterisation Of The Membrane Transport Of Pilocarpine In Cell Suspension Cultures Of Pilocarpus Microphyllus.

Pilocarpine is an alkaloid obtained from the leaves of Pilocarpus genus, with important pharmaceutical applications. Previous reports have investigated the production of pilocarpine by Pilocarpus microphyllus cell cultures and tried to establish the alkaloid biosynthetic route. However, the site of pilocarpine accumulation inside of the cell and its exchange to the medium culture is still unknown. Therefore, the aim of this study was to determine the intracellular accumulation of pilocarpine and characterise its transport across membranes in cell suspension cultures of P. microphyllus. Histochemical analysis and toxicity assays indicated that pilocarpine is most likely stored in the vacuoles probably to avoid cell toxicity. Assays with exogenous pilocarpine supplementation to the culture medium showed that the alkaloid is promptly uptaken but it is rapidly metabolised. Treatment with specific ABC protein transporter inhibitors and substances that disturb the activity of secondary active transporters suppressed pilocarpine uptake and release suggesting that both proteins may participate in the traffic of pilocarpine to inside and outside of the cells. As bafilomicin A1, a specific V-type ATPase inhibitor, had little effect and NH4Cl (induces membrane proton gradient dissipation) had moderate effect, while cyclosporin A and nifedipine (ABC proteins inhibitors) strongly inhibited the transport of pilocarpine, it is believed that ABC proteins play a major role in the alkaloid transport across membranes but it is not the exclusive one. Kinetic studies supported these results.

Effect of the toxic milk mutation (tx) on the function and intracellular localization of Wnd, the murine homologue of the Wilson copperATPase

Wilson disease is an autosomal recessive copper transport disorder resulting from defective biliary excretion of copper and subsequent hepatic copper accumulation and liver failure if not treated. The disease is caused by mutations in the ATP7B (WND) gene, which is expressed predominantly in the liver and encodes a copper-transporting P-type ATPase that is structurally and functionally similar to the Menkes protein (MNK), which is defective in the X-linked copper transport disorder Menkes disease. The toxic milk (tx) mouse has a clinical phenotype similar to Wilson disease patients and, recently, the tx mutation within the murine WND homologue (Wnd) of this mouse was identified, establishing it as an animal model for Wilson disease. In this study, cDNA constructs encoding the wild-type (Wnd-wt) and mutant (Wnd-tx) Wilson proteins (Wnd) were generated and expressed in Chinese hamster ovary (CHO) cells. The fx mutation disrupted the copper-induced relocalization of Wnd in CHO cells and abrogated Wnd-mediated copper resistance of transfected CHO cells. In addition, co-localization experiments demonstrated that while Wnd and MNK are located in the trans-Golgi network in basal copper conditions, with elevated copper, these proteins are sorted to different destinations within the same cell, Ultrastructural studies showed that with elevated copper levels, Wnd accumulated in large multivesicular structures resembling late endosomes that may represent a novel compartment for copper transport. The data presented provide further support for a relationship between copper transport activity and the copper-induced relocalization response of mammalian copper ATPases, and an explanation at a molecular level for the observed phenotype of fx mice.

Active transport of proton and calcium in higher plant cells.

Membrane transport of proton and calcium (Ca2+) plays a fundamental role in growth and developmental processes in higher plant cells. The plasma membrane contains an ATPase (P-ATPase) that pumps protons into the extracellular space, whereas two proton pumps, a vacuolar-type ATPase (V-ATPase) and a pyrophosphatase (H+-PPase) are associated with the tonoplast and pump protons into the vacuole. The P-ATPase, V-ATPase and H+-PPase catalyse electrogenic H+-translocation, giving rise to a proton motive force used to transport different molecules, via specific transport proteins (channels or carriers: H+-symport or H+-antiport), across the plasma membrane and the tonoplast

Calcineurin A of Candida albicans: involvement in antifungal tolerance, cell morphogenesis and virulence.

The azole antifungal fluconazole possesses only fungistatic activity in Candida albicans and, therefore, this human pathogen is tolerant to this agent. However, tolerance to fluconazole can be inhibited when C. albicans is exposed to fluconazole combined with the immunosuppressive drug cyclosporin A, which is known to inhibit calcineurin activity in yeast. A mutant lacking both alleles of a gene encoding the calcineurin A subunit (CNA) lost viability in the presence of fluconazole, thus making calcineurin essential for fluconazole tolerance. Consistent with this observation, tolerance to fluconazole was modulated by calcium ions or by the expression of a calcineurin A derivative autoactivated by the removal of its C-terminal inhibitory domain. Interestingly, CNA was also essential for tolerance to other antifungal agents (voriconazole, itraconazole, terbinafine, amorolfine) and to several other metabolic inhibitors (caffeine, brefeldin A, mycophenolic acid, fluphenazine) or cell wall-perturbing agents (SDS, calcofluor white, Congo red), thus indicating that the calcineurin pathway plays an important role in the survival of C. albicans in the presence of external growth inhibitors. Several genes, including PMC1, a vacuolar calcium P-type ATPase, were regulated in a calcineurin- and fluconazole-dependent manner. However, PMC1 did not play a direct role in the survival of C. albicans when exposed to fluconazole. In addition to these different properties, calcineurin was found to affect colony morphology in several media known to modulate the C. albicans dimorphic switch. In particular, calcineurin was found to be essential for C. albicans viability in serum-containing media. Finally, calcineurin was found to be necessary for the virulence of C. albicans in a mice model of infection, thus making calcineurin an important element for adequate adaptation to the conditions of the host environment.

Human PEX1 cloned by functional complementation on a CHO cell mutant is responsible for peroxisome-deficient Zellweger syndrome of complementation group I

The peroxisome biogenesis disorders (PBDs), including Zellweger syndrome (ZS) and neonatal adrenoleukodystrophy (NALD), are autosomal recessive diseases caused by defects in peroxisome assembly, for which at least 10 complementation groups have been reported. We have isolated a human PEX1 cDNA (HsPEX1) by functional complementation of peroxisome deficiency of a mutant Chinese hamster ovary (CHO) cell line, ZP107, transformed with peroxisome targeting signal type 1-tagged “enhanced” green fluorescent protein. This cDNA encodes a hydrophilic protein (Pex1p) comprising 1,283 amino acids, with high homology to the AAA-type ATPase family. A stable transformant of ZP107 with HsPEX1 was morphologically and biochemically restored for peroxisome biogenesis. HsPEX1 expression restored peroxisomal protein import in fibroblasts from three patients with ZS and NALD of complementation group I (CG-I), which is the highest-incidence PBD. A CG-I ZS patient (PBDE-04) possessed compound heterozygous, inactivating mutations: a missense point mutation resulting in Leu-664 → Pro and a deletion of the sequence from Gly-634 to His-690 presumably caused by missplicing (splice site mutation). Both PBDE-04 PEX1 cDNAs were defective in peroxisome-restoring activity when expressed in the patient fibroblasts as well as in ZP107 cells. These results demonstrate that PEX1 is the causative gene for CG-I peroxisomal disorders.

Localization of the Wilson’s disease protein product to mitochondria

Wilson’s disease (WND) is an inherited disorder of copper homeostasis characterized by abnormal accumulation of copper in several tissues, particularly in the liver, brain, and kidney. The disease-associated gene encodes a copper-transporting P-type ATPase, the WND protein, the subcellular location of which could be regulated by copper. We demonstrate that the WND protein is present in cells in two forms, the 160-kDa and the 140-kDa products. The 160-kDa product was earlier shown to be targeted to trans-Golgi network. The 140-kDa product identified herein is located in mitochondria as evidenced by the immunofluorescent staining of HepG2 cells with specific mitochondria markers and polyclonal antibody directed against the C terminus of the WND molecule. The mitochondrial location for the 140-kDa WND product was confirmed by membrane fractionation and by analysis of purified human mitochondria. The antibody raised against a repetitive sequence in the N-terminal portion of the WND molecule detects an additional 16-kDa protein, suggesting that the 140-kDa product was formed after proteolytic cleavage of the full-length WND protein at the N terminus. Thus, the WND protein is a P-type ATPase with an unusual subcellular localization. The mitochondria targeting of the WND protein suggests its important role for copper-dependent processes taking place in this organelle.